Acridone derivatives as labels for fluorescence detection of target materials

ABSTRACT

Disclosed are new acridone dye derivatives having characteristic fluorescence lifetimes. Also disclosed are methods for labelling target biological materials employing the acridone dyes and use of the labelled materials in biological assays. The acridone derivatives have the following structure:in which Z 1  and Z 2  represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 2 , R 3 , R 4  and R 5  are selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl, sulphonate, sulphonic acid, quatemary ammonium, the group —E—F and the group —(CH 2 —) n Y; R 1  is selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20  alkyl, aralkyl, the group —E—F and the group —(CH 2 —) n Y; where E is a spacer group, F is a target bonding group; Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6. The invention also relates to a set of different fluorescent acridone dye derivatives, each dye having a different fluorescence lifetime, the set of dyes being particularly useful for multiparameter analysis.

[0001] The present invention relates to new acridone derivatives havingcharacteristic fluorescence lifetimes that can be used as labels forattachment to and labelling of target materials. The acridonederivatives of the invention may be easily distinguished, one from theother, by virtue of their fluorescence lifetimes and they may be used inmultiparameter applications. The invention also relates to assay methodsutilising acridone derivatives and to a set of different fluorescentacridone lifetime dyes.

[0002] There is an increasing interest in, and demand for, fluorescentlabels for use in the labelling and detection of biological materials.Fluorescent labels are generally stable, sensitive and a wide range ofmethods are now available for the labelling of biomolecules. Typically,the emission spectrum of a fluorescent dye is a characteristic propertyof the dye, the intensity of such emission being used in the detectionof materials labelled with that dye. One problem with measurements offluorescence intensity as a means of detecting and/or measuring theconcentration of a fluorescent labelled biomolecule is that backgroundfluorescence may interfere with the measurement. Thus, in order toobtain improvements in the sensitivity of fluorescence detection, it ishighly desirable to improve the signal-to-noise ratio.

[0003] One means of overcoming the problem of background noise has beenthrough the use of long wavelength dyes, for example, the cyanine dyesCy™5 and Cy7, as disclosed in U.S. Pat. No. 5,268,486 (Waggoner et al).These dyes emit in the 600-800 nm region of the spectrum, wherebackground fluorescence is much less of a problem. Another means ofimproving the signal-to-noise ratio in fluorescence measurements is inthe use of time-resolved fluorescence, for example by using fluorescentlabels based on lanthanide chelates, eg. Eu³⁺ and Tb³⁺ (Selvin et al,U.S. Pat. No. 5,622,821). In time-resolved fluorescent labels, thelifetime of the fluorescence emission is typically longer than that ofthe background fluorescence, which may therefore be gated out usingappropriate instrumentation.

[0004] McGown, L. B. et al (Anal. Chem., (2000), 72, 5865-73) describethe use of a range of different dyes for multiparameter analysis inwhich fluorescence lifetime, rather than fluorescence wavelength, is thediscriminating characteristic. Dyes from different dye classes were usedto obtain lifetime resolution; however compensation was required foreither mobility differences or different fluorescence signalintensities. The method has been refined by Sauer, M. et al(J.Fluorescence, (1993), 3(3), 131-139) who employed a series ofrhodamine-based fluors having a range of fluorescent lifetimes but whichall absorb and emit at similar wavelengths, thus avoiding having tochange the excitation source and emission filters.

[0005] The acridone chromophore is highly fluorescent and has been usedfor labelling biological molecules and subsequent detection byconventional fluorescence emission spectroscopy. For example, Faller, T.et al (J.Chem.Soc.Chem.Comm., (1997), 1529-30) describe the preparationof a succinimidyl ester derivative of acridone and its use in labellingpeptides for subsequent analysis by mass-spectroscopy. U.S. Pat. No.5,472,582 (Jackson) describes the use of the fluorescent label,2-aminoacridone, for labelling and detecting carbohydrates in a mixture,following electrophoretic separation.

[0006] Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1978), 240(4), 884-7)have measured the fluorescence lifetimes of several acridonederivatives, however, to date, there appear to be no reports relating tothe use of acridones as lifetime dyes suitable for labelling and thedetection of biological materials. The present invention thereforedescribes modifications of the acridone chromophore, to produce a rangeof acridone derivatives having characteristic fluorescence lifetimes andwhich are useful for labelling biological materials.

[0007] The acridone derivatives of the present invention moreoverprovide a valuable set of fluorescent labels having a common corestructure and which are particularly useful for multiparameter analysis.In each dye of a set of dyes, the absorption and emission spectra remainessentially the same, whilst the fluorescence lifetimes vary. Thus, itis possible to use a common excitation source and determine thelifetimes at the same emission wavelength, thereby simplifyingrequirements for detection instrumentation used in multiparameterexperiments. Another advantage of the present invention is that thefluorescence lifetimes of the acridone dye derivatives are generallylonger than the lifetimes of other fluorescent labels, as well asnaturally occurring fluorescent materials, such as proteins andpolynucleotides, thereby allowing easy discrimination from backgroundfluorescence in biological assays utilising such dyes.

[0008] Accordingly, in a first aspect of the present invention there isprovided use of a reagent for labelling and lifetime detection of atarget material, wherein said reagent is a dye of the formula (I):

[0009] wherein:

[0010] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure;

[0011] Z¹ and Z² independently represent the atoms necessary to completeone ring, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur;

[0012] R², R³, R⁴ and R⁵ are independently selected from hydrogen,halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substitutedbenzyl, amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl,carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl,heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,quaternary ammonium, the group —E—F and the group —(CH₂—)_(n)Y;

[0013] R¹ is selected from hydrogen, mono- or di-nitro-substitutedbenzyl, C₁-C₂₀ alkyl, aralkyl, the group —E—F and the group—(CH₂—)_(n)Y;

[0014] E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0015] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6.

[0016] In a first embodiment of the first aspect, the dye of formula (I)is a fluorescent dye wherein:

[0017] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;

[0018] R², R³, R⁴ and R⁵ are independently selected from hydrogen,halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y; and

[0019] R¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group—E—F and the group —(CH₂—)_(n)Y;

[0020] wherein E, F, Y and n are hereinbefore defined.

[0021] The acridone dyes according to the first embodiment of the firstaspect are particularly suitable for use as fluorescence lifetime dyes.In the context of the present invention, the term lifetime dye isintended to mean a dye having a measurable fluorescence lifetime,defined as the average amount of time that the dye remains in itsexcited state following excitation (Lackowicz, J. R., Principles ofFluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, New York,(1999)).

[0022] Preferably, the fluorescent dye has a fluorescence lifetime inthe range from 2 to 30 nanoseconds, more preferably from 2 to 20nanoseconds.

[0023] In a second embodiment of the first aspect, the dye of formula(I) is a non-fluorescent or substantially non-fluorescent dye wherein:groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined; andwherein at least one of groups R, R², R³, R⁴ and R⁵ comprises at leastone nitro group.

[0024] In this embodiment, suitably, the at least one nitro group may beattached directly to the Z¹ and/or Z² ring structures. In thealternative, a mono- or di-nitro-substituted benzyl group may beattached to the R¹, R², R³, R⁴ or R⁵ positions, which optionally may befurther substituted with one or more nitro groups attached directly tothe Z¹ and/or Z² ring structures.

[0025] Preferably, in the first and second embodiments, at least one ofgroups R¹, R², R³, R⁴ and R⁵ in the dye of formula (I) is the group —E—Fwhere E and F are hereinbefore defined.

[0026] Suitably, the target bonding group F is a reactive or functionalgroup. A reactive group of a compound according to formula (I) can reactunder suitable conditions with a functional group of a target material;a functional group of a compound according to formula (I) can reactunder suitable conditions with a reactive group of the target materialsuch that the target material becomes labelled with the compound.

[0027] Preferably, when F is a reactive group, it is selected fromsuccinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,maleimide, haloacetamide, acid halide, vinylsulphone, dichlorotriazine,carbodiimide, hydrazide and phosphoramidite. Preferably, when F is afunctional group, it is selected from hydroxy, amino, sulphydryl,imidazole, carbonyl including aldehyde and ketone, phosphate andthiophosphate. By virtue of these reactive and functional groups thecompounds of formula (I) may be reacted with and covalently bond totarget materials.

[0028] Suitably, Z¹ and Z² may be selected independently from the groupconsisting of phenyl, pyridinyl, naphthyl, anthranyl, indenyl,fluorenyl, quinolinyl, indolyl, benzothiophenyl, benzofuranyl andbenzimidazolyl moieties. Additional one, two fused, or three fused ringsystems will be readily apparent to the skilled person. Preferably, Z¹and Z² are selected from the group consisting of phenyl, pyridinyl,naphthyl, quinolinyl and indolyl moieties. Particularly preferred Z¹ andZ² are phenyl and naphthyl moieties.

[0029] Preferably, at least one of the groups R¹, R², R³, R⁴ and R⁵ ofthe dyes of formula (I) is a water solubilising group for conferring ahydrophilic characteristic to the compound. Solubilising groups, forexample, sulphonate, sulphonic acid and quaternary ammonium, may beattached directly to the aromatic ring structures Z¹ and/or Z² of thecompound of formula (I). Alternatively, solubilising groups may beattached by means of a C₁ to C₆ alkyl linker chain to said aromatic ringstructures and may be selected from the group —(CH₂—)_(n)Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6. Alternativesolubilising groups may be carbohydrate residues, for example,monosaccharides. Examples of water solubilising constituents includeC₁-C₆ alkyl sulphonates, such as —(CH₂)₃—SO₃ ⁻ and —(CH₂)₄—SO₃ ⁻.However, one or more sulphonate or sulphonic acid groups attacheddirectly to the aromatic ring structures of a dye of formula (I) areparticularly preferred. Water solubility may be advantageous whenlabelling proteins.

[0030] Suitable spacer groups E may contain 1-60 chain atoms selectedfrom the group consisting of carbon, nitrogen, oxygen, sulphur andphosphorus. For example the spacer group may be:

[0031] —(CHR′)_(p)—

[0032] —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—

[0033] —{(CHR′)_(q)—S—(CHR′)_(r)}_(s)—

[0034] —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—

[0035] —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—

[0036] —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—

[0037] —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—

[0038] —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—

[0039] where R¹ is hydrogen, C₁-C₄ alkyl or aryl, which may beoptionally substituted with sulphonate, Ar is phenylene, optionallysubstituted with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is1-10 and s is 1-5.

[0040] Specific examples of reactive groups R¹, R², R³, R⁴ and R⁵ andthe groups with which R¹, R², R³, R⁴ and R⁵ can react are provided inTable 1. In the alternative, groups R¹, R², R³, R⁴ and R⁵ may be thefunctional groups of Table 1 that would react with the reactive groupsof a target material: TABLE 1 Possible Reactive Substituents and SitesReactive Therewith Reactive Groups Functional Groups succinimidyl estersprimary amino, secondary amino isothiocyanates amino groupshaloacetamides, maleimides sulphydryl, imidazole, hydroxyl, amine acidhalides amino groups anhydrides primary amino, secondary amino, hydroxylhydrazides, aldehydes, ketones vinylsulphones amino groupsdichlorotriazines amino groups carbodiimides carboxyl groupsphosphoramidites hydroxyl groups

[0041] Preferred reactive groups which are especially useful forlabelling target materials with available amino and hydroxyl functionalgroups include:

[0042] where n is 0 or an integer from 1-10.

[0043] Aryl is an aromatic substituent containing one or two fusedaromatic rings containing 6 to 10 carbon atoms, for example phenyl ornaphthyl, the aryl being optionally and independently substituted by oneor more substituents, for example halogen, hydroxyl, straight orbranched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl andC₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy and n-butoxy.

[0044] Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ringsystem containing at least one and no more than 3 heteroatoms which maybe selected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, hydroxyl,straight or branched chain alkyl groups containing 1 to 10 carbon atoms,aralkyl and C₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy andn-butoxy.

[0045] Aralkyl is a C₁ to C₆ alkyl group substituted by an aryl orheteroaryl group.

[0046] Halogen and halo groups are selected from fluorine, chlorine,bromine and iodine.

[0047] Exemplary dyes according to the first embodiment of the firstaspect are as follows:

[0048] i) O—(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate

[0049] ii)O—(N-succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate

[0050] iii)O—(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate

[0051] iv)O—(N-succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate.

[0052] The dyes of the present invention may be used to label andthereby impart fluorescent properties to a variety of target biologicalmaterials. Thus, in a second aspect, there is provided a method forlabelling a target biological material, the method comprising:

[0053] i) adding to a liquid containing said target biological materiala dye of formula (I):

[0054] wherein:

[0055] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;

[0056] R², R³, R⁴ and R¹ are independently selected from hydrogen,halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y;

[0057] R¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group—E—F and the group —(CH₂—)_(n)Y;

[0058] where E, F, Y and n are hereinbefore defined; and

[0059] ii) incubating said dye with said target biological materialunder conditions suitable for labelling said target.

[0060] Suitably, the fluorescent dyes of the present invention whereinat least one of the groups R¹ to R⁵ contains a charge, for example,quaternary amino, may be used to bind non-covalently to chargedbiological molecules such as, for example, DNA and RNA. Alternatively,fluorescent dyes of the present invention wherein at least one of thegroups R¹ to R⁵ is an uncharged group, for example, a long chain alkylor an aryl group, may be used to bind to uncharged biological moleculessuch as, for example, biological lipids, as well as to intact cellmembranes, membrane fragments and cells.

[0061] In a preferred embodiment, at least one of the groups R¹, R², R³,R⁴ and R⁵ in the dye of formula (I) is the group —E—F where E and F arehereinbefore defined. In this embodiment, the fluorescent dyes may beused to covalently label a target biological material. The targetbonding group may be a reactive group for reacting with a functionalgroup of the target material. Alternatively, the target bonding groupmay be a functional group for reacting with a reactive group on thetarget biological material. The method comprises incubating the targetmaterial with an amount of the dye according to the invention underconditions to form a covalent linkage between the target and the dye.The target may be incubated with an amount of a compound according tothe present invention having at least one of groups R¹, R², R³, R⁴ andR⁵ that includes a reactive or functional group as hereinbefore definedthat can covalently bind with the functional or reactive group of thetarget biological material.

[0062] Suitable biological materials include, but are not limited to thegroup consisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs, hormones, cells, cell membranes and toxins.

[0063] The fluorescent dyes according to the invention having a targetbonding group in at least one of groups R¹, R², R³, R⁴ and R⁵ may beused in an assay method for determining the presence or the amount of ananalyte in a sample. Thus, in a third aspect of the present invention,there is provided a method for the assay of an analyte in a sample whichmethod comprises:

[0064] i) contacting the analyte with a specific binding partner forsaid analyte under conditions suitable to cause the binding of at leasta portion of said analyte to said specific binding partner to form acomplex and wherein one of said analyte and said specific bindingpartner is labelled with a fluorescent dye of formula (I):

[0065] wherein:

[0066] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;

[0067] at least one of groups R¹, R², R³, R⁴ and R⁵ is the group —E—Fwhere E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0068] when any of said groups R², R³, R⁴ and R⁵ is not said group —E—F,said remaining groups R², R³, R⁴ and R⁵ are independently selected fromhydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium and the group—(CH₂—)_(n)Y; and, when group R¹ is not said group —E—F, it is selectedfrom hydrogen, C₁-C₂₀ alkyl, aralkyl and the group —(CH₂—)_(n)Y;

[0069] wherein Y and n are hereinbefore defined;

[0070] ii) measuring the emitted fluorescence of the labelled complex;and

[0071] iii) correlating the emitted fluorescence with the presence orthe amount of said analyte in said sample.

[0072] Suitably, step ii) may be performed by measurement of thefluorescence intensity or fluorescence lifetime of the sample,preferably the fluorescence lifetime.

[0073] In one embodiment, the assay method is a direct assay for themeasurement of an analyte in a sample. Optionally, a known or putativeinhibitor compound may be included in the assay mix.

[0074] In a second, or alternative embodiment, the assay may be acompetitive assay wherein a sample containing an analyte competes with afluorescent tracer for a limited number of binding sites on a bindingpartner that is capable of specifically binding the analyte and thetracer. Suitably, the tracer is a labelled analyte or a labelled analyteanalogue, in which the label is a fluorescent dye of formula (I).Increasing amounts (or concentrations) of the analyte in the sample willreduce the amount of the fluorescent labelled analyte or fluorescentlabelled analyte analogue that is bound to the specific binding partner.The fluorescence signal is measured and the concentration of analyte maybe obtained by interpolation from a standard curve.

[0075] In a further embodiment, the binding assay may employ a two-stepformat, wherein a first component (which may be optionally coupled to aninsoluble support) is bound to a second component to form a specificbinding complex, which is bound in turn to a third component. In thisformat, the third component is capable of specifically binding to eitherthe second component, or to the specific binding complex. Either of thesecond or the third component may be labelled with a fluorescent dyeaccording to the present invention. Examples include “sandwich” assays,in which one component of a specific binding pair, such as a firstantibody, is coated onto a surface, such as the wells of a multiwellplate. Following the binding of an antigen to the first antibody, afluorescent labelled second antibody is added to the assay mix, so as tobind with the antigen-first antibody complex. The fluorescence signal ismeasured and the concentration of antigen may be obtained byinterpolation from a standard curve.

[0076] Examples of analyte-specific binding partner pairs include, butare not restricted to, antibodies/antigens, lectins/glycoproteins,biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood thatany molecules which possess a specific binding affinity for each othermay be employed, so that the fluorescent dyes of the present inventionmay be used for labelling one component of a specific binding pair,which in turn may be used in the detection of binding to the othercomponent.

[0077] The fluorescent dyes according to first embodiment of the firstaspect may be used in applications that include detecting anddistinguishing between various components in a mixture. Thus, in afourth aspect, the present invention provides a set of two or moredifferent fluorescent dyes according to the invention, each dye of saidset of dyes having the formula (I):

[0078] wherein:

[0079] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure, where Z¹ and Z² arehereinbefore defined;

[0080] R², R³, R⁴ and R⁵ are independently selected from hydrogen,halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy,acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl,sulphonate, sulphonic acid, quaternary ammonium, the group —E—F and thegroup —(CH₂—)_(n)Y;

[0081] R¹ is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl, the group—E—F and the group —(CH₂—)_(n)Y;

[0082] E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group;

[0083] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;

[0084] wherein each dye of said set has a distinguishably differentfluorescence lifetime compared with the lifetimes of the remaining dyesof the set.

[0085] Preferably, in each dye of the set of dyes at least one of groupsR¹, R², R³, R⁴ and R⁵ is the group —E—F where E and F are hereinbeforedefined.

[0086] Preferably, the set of fluorescent dyes according to theinvention will comprise four different dyes, each dye of the set havinga different fluorescence lifetime.

[0087] Preferably, each of the fluorescent dyes in the set has afluorescence lifetime in the range from 2 to 30 nanoseconds. Morepreferably the fluorescent dyes in the set will have fluorescencelifetimes in the range from 2 to 20 nanoseconds.

[0088] To distinguish between different dyes in the set of dyes, thedifference in the lifetimes of the fluorescent emission of two such dyesis preferably at least 15% of the value of the shorter lifetime dye.

[0089] The set of dyes may be used in a detection method whereindifferent fluorescent dyes of the set of dyes are covalently bonded to aplurality of different primary components, each primary component beingspecific for a different secondary component, in order to identify eachof a plurality of secondary components in a mixture of secondarycomponents. The method comprises covalently binding different dyes of aset of fluorescent dyes according to the fourth aspect of the inventionto different primary components in a multicomponent mixture wherein eachdye of the set has a different fluorescence lifetime, compared with thefluorescence lifetimes of the remaining dyes of the set; adding thedye-labelled primary components to a preparation containing secondarycomponents under conditions to enable binding of at least a portion ofeach of said dye-labelled primary components to its respective secondarycomponent; and determining the presence or the amount of the boundsecondary component by measuring the fluorescence lifetime of each ofthe labelled primary component-secondary component complexes.

[0090] If required, any unreacted primary components may be removed orseparated from the preparation by, for example washing, to preventinterference with the analysis.

[0091] Preferably, a single wavelength of excitation can be used toexcite fluorescence from two or more materials in a mixture, where eachfluoresces having a different characteristic fluorescent lifetime.

[0092] The set of fluorescent dyes according to the present inventionmay be used in any system in which the creation of a fluorescent primarycomponent is possible. For example, an appropriately reactivefluorescent dye according to the invention can be conjugated to a DNA orRNA fragment and the resultant conjugate then caused to bind to acomplementary target strand of DNA or RNA. Other examples of primarycomponent-secondary component complexes which may be detected includeantibodies/antigens and biotin/streptavidin.

[0093] The set of dyes according to the present invention may also beadvantageously used in fluorescent DNA sequencing based uponfluorescence lifetime discrimination of the DNA fragments. Briefly, eachone of a set of dyes, may be coupled to a primer. Various primers areavailable, such as primers from pUC/M13, λgt10, λgt11 and the like (seeSambrook et al, Molecular Cloning, A Laboratory Manual 2^(nd) Edition,Cold Spring Harbour Laboratory Press 1989). DNA sequences are clonedinto an appropriate vector having a primer sequence joined to the DNAfragment to be sequenced. After hybridisation to the DNA template,polymerase enzyme-directed synthesis of a complementary strand occurs.Different 2′,3′-dideoxynucleotide terminators are employed in eachdifferent sequencing reaction so as to obtain base-specific terminationof the chain extension reaction. The resulting set of DNA fragments areseparated by electrophoresis and the terminating nucleotide (and thusthe DNA sequence) is determined by detecting the fluorescence lifetimeof the labelled fragments. DNA sequencing may also be performed usingdideoxynucleotide terminators covalently labelled with the fluorescentdyes according to the present invention.

[0094] The non-fluorescent or substantially non-fluorescent dyesaccording to the second embodiment of the first aspect may be used asthe substrate for an enzyme and which upon reaction with the enzyme,yields a fluorescent product.

[0095] Bacterial nitroreductases have been shown to catalyse the generalreaction set out below in Reaction Scheme 1.

[0096] where, in the presence of NADH or NADPH, one or more nitro groupson an organic molecule may be reduced to a hydroxylamine (—NHOH) groupwhich may subsequently be converted to an amine (—NH₂) group.

[0097] Thus, in a fifth aspect of the invention, there is provided amethod of increasing the fluorescence of a dye of formula (I):

[0098] wherein:

[0099] groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group;

[0100] characterised by the reduction of said at least one nitro groupto —NHOH or—NH₂

[0101] Preferably, the fluorescence lifetime of the fluorescent productof the reduction is in the range from 2 to 30 nanoseconds.

[0102] Suitably, reduction is by means of nitroreductase. This can beachieved by enzymatic conversion of a nitro group in a compound offormula (I) to a —NHOH or —NH₂ group by the action of thenitroreductase. Depending on the structure of the dye, the fluorescenceemission from the product of the nitroreductase reaction may typicallyhave a lifetime in the range 2 to 30 nanoseconds. Moreover, thefluorescence lifetime characteristics of the reaction product can bealtered to suit the application by means of additional substitutents,whilst retaining the nitro group(s) that are involved in the reactionwith nitroreductase. Thus, fluorescent reporters compatible for use withother fluors in multiplex systems can be provided.

[0103] In a sixth aspect of the invention there is provided a method fordetecting nitroreductase enzyme activity in a composition comprising:

[0104] i) mixing under conditions to promote nitroreductase activitysaid composition with a dye of formula (I):

[0105] wherein:

[0106] groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; and

[0107] ii) measuring an increase in fluorescence wherein said increaseis a measure of the amount of nitroreductase activity.

[0108] Suitably, the measurement of step ii) may be of the fluorescenceintensity and/or fluorescence lifetime of the dye.

[0109] In one embodiment of the sixth aspect, the composition comprisesa cell or cell extract. In principle, any type of cell can be used, i.e.prokaryotic or eukaryotic (including bacterial, mammalian and plantcells). Where appropriate, a cell extract can be prepared from a cell,using standard methods known to those skilled in the art (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press 1989), prior to measuring fluorescence.

[0110] Typical conditions for nitroreductase activity compriseincubation of the composition in a suitable medium and the dye atapproximately 37° C. in the presence of NADH and FMN.

[0111] In a seventh aspect of the invention there is provided an assaymethod comprising:

[0112] i) binding one component of a specific binding pair to a surface;

[0113] ii) adding a second component of the specific binding pair underconditions to promote binding between the components, said secondcomponent being labelled with a nitroreductase enzyme;

[0114] iii) adding a dye of formula (I):

[0115] wherein:

[0116] groups R¹, R² ₁ R³ ₁ R⁴, R⁵, Z¹ and Z² are hereinbefore definedand wherein at least one of groups R¹, R², R³, R⁴ and R¹ comprises atleast one nitro group; and

[0117] iv) detecting binding of the second component to the firstcomponent by measuring an increase in fluorescence as a measure of boundnitroreductase activity.

[0118] In a preferred embodiment of the seventh aspect, said specificbinding pair is selected from the group consisting ofantibodies/antigens, lectins/glycoproteins, biotin/streptavidin,hormone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA and DNA/bindingprotein.

[0119] Briefly, an in vitro assay method for the detection of antibodybinding may be configured as follows. An antibody specific for anantigen of interest may be labelled by covalently linking it to anenzymatically active nitroreductase. The labelled antibody can then beintroduced into the test sample containing the antigen under conditionssuitable for binding. After washing to remove any unbound antibody, theamount of bound antibody is detected by incubating the sample with asubstrate comprising a compound of formula (I) having at least one nitrogroup under conditions for nitroreductase activity and measuring anincrease in fluorescence. The amount of fluorescence detected will beproportional to the amount of nitroreductase-labelled antibody that hasbound to the antigen.

[0120] In an in vitro assay for detecting the binding of nucleic acidsby hybridisation, either of the pair of target and probe nucleic acid isimmobilised by attachment to a membrane or surface. The second member ofthe pair is labelled with nitroreductase and incubated under hybridisingconditions with the immobilised nucleic acid. Unbound, labelled nucleicacid is washed off and the amount of bound, labelled nucleic acid ismeasured by incubating the membrane or surface with a compound offormula (I) having at least one nitro group under conditions suitablefor nitroreductase activity. The amount of increase in fluorescencegives a measure of the amount of bound labelled DNA.

[0121] Methods for coupling enzymes, such as nitroreductase, to otherbiomolecules, e.g. proteins and nucleic acids, are well known(Bioconjugate Techniques, Academic Press 1996). Coupling may be achievedby direct means, for example by use of a suitable bifunctionalcrosslinking agent (e.g. N-[γ-maleimidopropionic acid]hydrazine, Pierce)to covalently link the enzyme and binding partner. Alternatively,coupling may be achieved by indirect means, for example by separatelybiotinylating the enzyme and the binding partner using a chemicallyreactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce)and subsequently coupling the molecules through a streptavidin bridgingmolecule.

[0122] Cell based assays are increasingly attractive over in vitrobiochemical assays for use in high throughput screening (HTS). This isbecause cell based assays require minimal manipulation and the readoutscan be examined in a biological context that more faithfully mimics thenormal physiological situation. Such in vivo assays require an abilityto measure a cellular process and a means to measure its output. Forexample, a change in the pattern of transcription of a number of genescan be induced by cellular signals triggered, for example, by theinteraction of an agonist with its cell surface receptor or by internalcellular events such as DNA damage. The induced changes in transcriptioncan be identified by fusing a reporter gene to a promoter region whichis known to be responsive to the specific activation signal.

[0123] In fluorescence-based enzyme-substrate systems, an increase influorescence gives a measure of the activation of the expression of thereporter gene.

[0124] Accordingly, in a eighth aspect of the invention, there isprovided an assay method which comprises:

[0125] i) contacting a host cell which has been transfected with anucleic acid molecule comprising expression control sequences operablylinked to a sequence encoding a nitroreductase, with a dye of formula(I):

[0126] wherein:

[0127] groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbefore defined andwherein at least one of groups R¹, R², R³, R⁴ and R⁵ comprises at leastone nitro group; and

[0128] ii) measuring an increase in fluorescence as a measure ofnitroreductase gene expression.

[0129] In one embodiment of the eighth aspect, the assay method isconducted in the presence of a test agent whose effect on geneexpression is to be determined.

[0130] Methods for using a variety of enzyme genes as reporter genes inmammalian cells are well known (for review see Naylor L. H., BiochemicalPharmacology, (1999), 58, 749-757). The reporter gene is chosen to allowthe product of the gene to be measurable in the presence of other iscellular proteins and is introduced into the cell under the control of achosen regulatory sequence which is responsive to changes in geneexpression in the host cell. Typical regulatory sequences include thoseresponsive to hormones, second messengers and other cellular control andsignalling factors. For example, agonist binding to seven transmembranereceptors is known to modulate promoter elements including the cAMPresponsive element, NF-AT, SRE and AP1; MAP kinase activation leads tomodulation of SRE leading to Fos and Jun transcription; DNA damage leadsto activation of transcription of DNA repair enzymes and the tumoursuppressor gene p53. By selection of an appropriate regulatory sequencethe reporter gene can be used to assay the effect of added agents oncellular processes involving the chosen regulatory sequence under study.

[0131] For use as a reporter gene, the nitroreductase gene may beisolated by well known methods, for example by amplification from a cDNAlibrary by use of the polymerase chain reaction (PCR) (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press (1989) pp 14.5-14.20). Once isolated, thenitroreductase gene may be inserted into a vector suitable for use withmammalian promoters (Molecular Cloning, A Laboratory Manual 2^(nd)Edition, Cold Spring Harbour Laboratory Press (1989) pp 16.56-16.57) inconjunction with and under the control of the gene regulatory sequenceunder study. The vector containing the nitroreductase reporter andassociated regulatory sequences may then be introduced into the hostcell by transfection using well known techniques, for example by use ofDEAE-Dextran or Calcium Phosphate (Molecular Cloning, A LaboratoryManual 2^(nd) Edition, Cold Spring Harbour Laboratory Press (1989) pp16.30-16.46). Other suitable techniques will be well known to thoseskilled in the art.

[0132] In another embodiment of the eighth aspect, the dye of formula(I) wherein groups R¹, R², R³, R⁴, R⁵, Z¹ and Z² are hereinbeforedefined and wherein at least one of groups R¹, R², R³, R⁴ and R⁵comprises at least one nitro group, is permeable to cells. In thisembodiment, preferably, at least one of groups R¹, R², R³, R⁴ or R⁵comprises a cell membrane permeabilising group. Membrane permeantcompounds can be generated by masking hydrophilic groups to provide morehydrophobic compounds. The masking groups can be designed to be cleavedfrom the substrate within the cell to generate the derived substrateintracellularly. Because the substrate is more hydrophilic than themembrane permeant derivative it is then trapped in the cell. Suitablecell membrane permeabilising groups may be selected from acetoxymethylester which is readily cleaved by endogenous mammalian intracellularesterases (Jansen, A. B. A. and Russell, T. J., J.Chem.Soc., (1965),2127-2132 and Daehne W. et al. J.Med.Chem., (1970) 13, 697-612) andpivaloyl ester (Madhu et al., J. Ocul.Pharmacol.Ther., (1998), 14(5),389-399) although other suitable groups will be recognised by thoseskilled in the art.

[0133] Typically, to assay the activity of a test agent to activatecellular responses via the regulatory sequence under study, cellstransfected with the nitroreductase reporter are incubated with the testagent, followed by addition of a dye of formula (I) wherein at least oneof groups R¹, R², R³, R⁴ and R⁵ in said dye comprises at least one nitrogroup, said compound being made cell permeant. After an appropriateperiod required for conversion of the substrate to a form exhibitingfluorescence, the fluorescence from the cells is measured at an emissionwavelength appropriate for the chosen dye. Measurement of fluorescencemay be readily achieved by use of a range of detection instrumentsincluding fluorescence microscopes (e.g. LSM 410, Zeiss), microplatereaders (e.g. CytoFluor 4000, Perkin Elmer), CCD imaging systems (e.g.LEADseeker™, Amersham Pharmacia Biotech) and Flow Cytometers (e.g.FACScalibur, Becton Dickinson).

[0134] The measured fluorescence is compared with fluorescence fromcontrol cells not exposed to the test agent and the effects, if any, ofthe test agent on gene expression modulated through the regulatorysequence, is determined by the detection of the characteristicfluorescence in the test cells. Where appropriate, a cell extract can beprepared using conventional methods.

[0135] Suitable means for expressing a nitroreductase enzyme include anexpression plasmid or other expression construct. Methods for preparingsuch expression constructs are well known to those skilled in the art.

[0136] In an ninth aspect of the present invention, there is provided adye of formula (I):

[0137] wherein:

[0138] groups R² and R³ are attached to the Z¹ ring structure and groupsR⁴ and R⁵ are attached to the Z² ring structure;

[0139] Z¹ and Z² independently represent the atoms necessary to completeone ring, two fused ring, or three fused ring aromatic or heteroaromaticsystems, each ring having five or six atoms selected from carbon atomsand optionally no more than two atoms selected from oxygen, nitrogen andsulphur;

[0140] at least one of groups R¹, R², R³, R⁴ and R⁵ is the group —E—Fwhere E is a spacer group having a chain from 1-60 atoms selected fromthe group consisting of carbon, nitrogen, oxygen, sulphur and phosphorusatoms and F is a target bonding group; and,

[0141] when any of said groups R¹, R², R³, R⁴ and R⁵ is not said group—E—F, said remaining groups R², R³, R⁴ and R⁵ are independently selectedfrom hydrogen, halogen, amide, hydroxyl, cyano, nitro, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium and the group—(CH₂—)_(n)Y; and,

[0142] when group R¹ is not said group —E—F, it is selected fromhydrogen, mono- or di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyland the group —(CH₂—)_(n)Y; E is a spacer group having a chain from 1-60atoms selected from the group consisting of carbon, nitrogen, oxygen,sulphur and phosphorus atoms and F is a target bonding group;

[0143] Y is selected from sulphonate, sulphate, phosphonate, phosphate,quaternary ammonium and carboxyl; and n is an integer from 1 to 6;provided that at least one of groups R¹, R², R³, R⁴ and R⁵ is a watersolubilising group.

[0144] Preferably, the target bonding group F comprises a reactive groupfor reacting with a functional group on a target material, or afunctional group for reacting with a reactive group on a targetmaterial. Preferred reactive groups may be selected from carboxyl,succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate,maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone,dichlorotriazine and phosphoramidite. Preferred functional groups may beselected from hydroxy, amino, sulphydryl, imidazole, carbonyl includingaldehyde and ketone, phosphate and thiophosphate.

[0145] Preferably, the spacer group E is selected from:

[0146] —(CHR′)_(p)—

[0147] —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—

[0148] —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—

[0149] —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—

[0150] —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—

[0151] —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—

[0152] —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—

[0153] where R¹ is hydrogen, C₁-C₄ alkyl or aryl, which may beoptionally substituted with sulphonate, Ar is phenylene, optionallysubstituted with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is1-10 and s is 1-5.

[0154] The dyes of formula (I) may be prepared from the correspondingdiphenylamine-2-carboxylic acid according to published methods (seeAlbert, A. and Ritchie, B., Org. Syntheses, (1942), 22, 5; also U.S.Pat. No. 3,021,334). Suitably, the diphenylamine-2-carboxylic acid maybe heated in the presence of an acidic dehydrating agent such asphosphorus oxychloride or concentrated sulfuric acid. Thediphenylamine-2-carboxylic acid derivatives may be prepared by reactionof a 2-halobenzoic acid with a suitable primary aminobenzene (having atleast one aryl ring position unsubstituted ortho- to the amino group),which reaction may be performed in the presence of catalytic coppermetal/salt (see Ullmann, F., Chem. Ber., (1903), 36, 2382; also BritishPat. 649197). Suitably, the 2-halobenzoic acid is heated with theaminobenzene, in the presence of a base such as an alkali metalcarbonate, in a solvent such as 1-butanol or 1-pentanol. A catalyticamount of copper metal powder or a copper salt such as anhydrous copperacetate is also usually included, although sometimes this is notrequired.

[0155] It will be readily appreciated that certain dyes of the presentinvention may be useful as intermediates for conversion to other dyes bymethods well known to those skilled in the art. The dyes of the presentinvention may be synthesized by the methods disclosed herein.Derivatives of the compounds having a particular utility are preparedeither by selecting appropriate precursors or by modifying the resultantcompounds by known methods to include functional groups at a variety ofpositions. As examples, the dyes of the present invention may bemodified to include certain reactive groups for preparing a dyeaccording to the present invention, or charged or polar groups may beadded to enhance the solubility of the compound in polar or nonpolarsolvents or materials. As examples of conversions, an ester may beconverted to a carboxylic acid or may be converted to an amidoderivative. Groups R₁ to R⁵ may be chosen so that the dyes of thepresent invention have different lifetime characteristics, therebyproviding a number of related dyes which can be used in multiparameteranalyses wherein the presence and quantity of different compounds in asingle sample may be differentiated based on the wavelengths andlifetimes of a number of detected fluorescence emissions. The dyes ofthe present invention may be made soluble in aqueous, other polar, ornon-polar media containing the material to be labelled by appropriateselection of R-groups.

[0156] The invention is further illustrated by reference to thefollowing examples and figures in which:

[0157]FIG. 1 shows the absorbance spectra (1A) and the emission spectra(1B) of four acridone dyes according to the present invention;

[0158]FIG. 2 shows the fluorescence lifetime decay plot of four acridonedyes according to the present invention, as follows:

[0159] ______ : N-(succinyl)-2-amino-10H-acridine-9-one;

[0160] . . . . . . : 6-(9-oxo-9H-acridin-10-yl)hexanoic acid;

[0161] - - - - : 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid;

[0162] . . _ . . _ : 6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid;

[0163]FIG. 3 shows lifetime decay plots of protein conjugates asfollows: Conjugate 1=6-(9-oxo-9H-acridin-10-yl)hexanoic acid—bovineserum albumin (BSA) conjugate; Conjugate 2=6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid-rabbit serum albuminconjugate;

[0164]FIG. 4 is a lifetime decay plot following immunoprecipitation withanti-BSA antibody as described in Example 16;

[0165]FIG. 5 illustrates fluorescence lifetime detection in capillaryelectrophoresis of four acridone dye-labelled DNA fragments as describedin Example 17;

[0166]FIG. 6 shows the lifetime detection of a mixture of two differentacridone dye-labelled BSA conjugates and co-electrophoresed in SDS PAGEas described in Example 19.

[0167] Cy™ is a trademark of Amersham Biosciences UK Limited.

EXAMPLES 1. O—(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate

[0168]

[0169] 1.1 O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate

[0170] 9-(10H)-Acridone (4.88 g, 25 mmol) was mixed with anhydrousmethyl sulfoxide (25 ml) under nitrogen atmosphere and set stirring.After 5 minutes, the resultant yellow slurry was treated with potassiumtert-butoxide (3.37 g, 30 mmol) and stirring continued until all thesolids had dissolved. Ethyl 6-bromohexanoate (6.7 g, 30 mmol) was thenadded and the resulting solution stirred under nitrogen for 3 days. Atthe end of this time the reaction mixture was poured into water (1000ml) and extracted with ethyl acetate. The organic solution was washedwith 0.5M aqueous HCl, then with water, before being dried (MgSO₄),filtered and evaporated under vacuum.

[0171] The crude product was separated from unreacted acridone bytrituration with 1:1 ethyl acetate/hexane; acridone remained undissolvedand was filtered off. The filtrate was washed twice with 0.5M aqueousHCl before being dried (MgSO₄), filtered and evaporated under vacuum;the acid wash removed most of the O-alkylated acridine side product. Theresidue was then subjected to flash column chromatography (silica.30-50% ethyl acetate/hexane) to give 5.9 g (70%) ofO-ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate. This material was finallyrecrystallized from ethanol (20 ml) to give 5.14 g of analytically purematerial. λ_(max) (EtOH)=404, 387, 254 nm; δ_(H) (300 MHz, CD₃OD) 1.23(3H, t), 1.60 (2H, m), 1.73 (2H, m), 1.92 (2H, m), 2.37 (2H, t), 4.11(2H, q), 4.50 (2H, app.t) 7.34 (2H, m), 7.77 (2H, m), 7.84 (2H, m) and8.46 (2H, m). Mass spectrum: (ES+) 338 (M+H), 360 (M+Na).

[0172] 1.2 6-(9-Oxo-9H-acridin-10-yl)hexanoic acid

[0173] O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (3.40 g, 10 mmol) wasmixed with acetic acid (40 ml) and 1.0M aqueous HCl (10 ml). Theresulting solution was heated under reflux for 3 hrs until TLC indicatedcomplete conversion to the carboxylic acid (RPC₁₈. Methanol, 90:water,10. R_(f)=0.6). The solution was evaporated under vacuum, thenco-evaporated with acetonitrile until a yellow solid was obtained. Thiswas triturated with diethyl ether and dried under high vacuum overphosphorus pentoxide to give 6-(9-oxo-9H-acridin-10-yl)hexanoic acid(3.07 g, 98%). λ_(max) (EtOH)=404, 384, 256 nm. δ_(H) (300 MHz, CD₃OD)1.62 (2H, m), 1.75 (2H, m), 1.95 (2H, m), 2.36 (2H, t), 4.53 (2H,app.t), 7.35 (2H, m), 7.79 (2H, m), 7.86 (2H, m) and 8.46 (2H, m). Massspectrum: (ES+) 310 (M+H), 332 (M+Na). Melting Point=167° C.

[0174] 1.3 0-(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate

[0175] 6-(9-Oxo-9H-acridin-10-yl)hexanoic acid (309 mg, 1.0 mmol) andO-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU;301 mg, 1.0 mmol) were dissolved in N,N-dimethylformamide (5 ml). To theresulting solution was added N,N-diisopropylethylamine (183 μl, 1.05mmol). After 2 hrs the solvent was evaporated under vacuum. The residuewas purified by flash chromatography (silica. 0-10% ethylacetate/dichloromethane) to giveO-(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate as a pale yellowpowder (330 mg, 81%). δ_(H) (200 MHz, DMSO-d₆) 1.63-1.83 (6H, m), 2.74(2H, t), 2.83 (4H, s), 4.47 (2H, app.t), 7.30-7.38 (2H, m), 7.81-7.84(4H, m) and 8.34-8.38 (2H, m). Mass spectrum: (ES+) 407 (M+H), 429(M+Na). Accurate mass: (M+H)=C₂₃H₂₃N₂O₅, requires 407.1607. Found407.1597 (−2.4 ppm).

2. O-(N-Succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate

[0176]

[0177] 2.1 O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate andO-ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

[0178] O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (4.22 g, 12.5 mmol)was dissolved in ethanol (150 ml) with stirring. To the resultingsolution was added benzyltrimethylammoniun tribromide (9.75 g, 25 mmol).The mixture was stirred under nitrogen for 4 days. The solvent was thenevaporated under vacuum and the residue partitioned between water (1000ml) and ethyl acetate (300 ml). The organic layer was collected, washedwith more water and dilute aqueous sodium thiosulfate solution, thendried (MgSO₄), filtered and evaporated under vacuum.

[0179] The crude product was purified by flash column chromatography(silica. 0-5% ethyl acetate/dichloromethane).O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate eluted first,followed by O-ethyl-6-2-bromo-9-oxo-9H-acridin-10-yl)hexanoate. Purefractions of each were pooled and evaporated separately.

[0180] O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate wasrecrystallized from ethanol. Yield: 2.77 g (53%). λ_(max) (EtOH)=412,392, 300, 278, 256 nm. δ_(H) (300 MHz, CD₃OD) 1.22 (3H, t), 1.58 (2H,m), 1.74 (2H, m), 1.91 (2H, m), 2.37 (2H, t), 4.10 (2H, q), 4.49 (2H,app.t), 7.36 (1H, m), 7.72 (1H, d), 7.78 (1H, d), 7.82-7.92 (2H, m),8.42 (1H, dd) and 8.52 (1H, dd). Mass spectrum: (ES+) 416 and 418 (M+H),438 and 440 (M+Na).

[0181] O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate wasrecrystallized from chloroform/ethanol. Yield: 2.06 g (33%). δ_(H) (200MHz, DMSO-d₆) 1.15 (3H, t), 1.50-1.80 (6H, m), 2.31 (2H, t), 4.04 (2H,q), 4.43 (2H, app.t), 7.81 (2H, d), 7.95 (2H, dd) and 8.34 (2H, d).

[0182] 2.2 6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic acid

[0183] O-Ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (2.5 g, 6mmol) was dissolved in acetic acid (30 ml). To this solution was added1.0M aqueous HCl (10 ml). The mixture was heated under reflux for 3.5hrs. Reverse phase chromatographic analysis (C₁₈) (methanol:water,90:10) indicated two spots at R_(f)=0.3 and R_(f)=0.55. The solution wasthen evaporated under vacuum, then co-evaporated with acetonitrile untila yellow solid was obtained. This was triturated with diethyl ether anddried under high vacuum over phosphorus pentoxide to give6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid (2.27 g, 97%). δ_(H)(200 MHz, DMSO-d₆) 1.50-1.65 (4H, m), 1.70-1.81 (2H, m), 2.25 (2H, t),4.46 (2H, app.t), 7.32-7.40 (1H, m), 7.78-7.98 (4H, m) and 8.31-8.41(2H, m). λ_(max) (EtOH)=414, 397, 256 nm. Melting Point=213° C.

[0184] 2.3O-(N-Succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate

[0185] 6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic acid (388 mg, 1.0mmol) and O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TSTU; 301 mg, 1.0 mmol) were dissolved inN,N-dimethylformamide (5 ml). To the resulting solution was addedN,N-diisopropy(ethylamine (183 μl, 1.05 mmol). After 2 hrs the solventwas evaporated under vacuum. The residue was purified by flashchromatography (silica. 0-10% ethyl acetate/dichloromethane) to giveO-(N-succinimidyl)-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (330 mg,87%) δ_(H) (200 MHz, DMSO-d₆) 1.5-1.8 (6H, m), 2.73 (2H, t), 2.80 (4H,s), 4.47 (2H, app.t), 7.32-7.42 (1H, m), 7.78-7.83 (3H, m), 7.94 (1H,dd), 8.35 (1H, d) and 8.41 (1H, d). Mass spectrum: (ES+) 485+487 (M+H),507/509 (M+Na). Accurate mass: (M+H)=C₂₃H₂₂BrN₂O₅, requires 485.0712.Found 485.0689 (−4.8 ppm).

3. O-(N-Succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

[0186]

[0187] 3.1 6-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid

[0188] O-Ethyl-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate (2.0 g,4.04 mmol) was dissolved in acetic acid (30 ml). To this solution wasadded 1.0M aqueous HCl (10 ml). The mixture was heated under reflux for4 hrs. Reverse phase chromatographic analysis (C₁₈) (methanol:water,90:10) indicated two spots at R_(f)=0.2 and R_(f)=0.4. The solution wasallowed to cool to ambient temperature, whereupon the productcrystallized as fluffy yellow needles. After final cooling in an icebath, the solid was collected by vacuum filtration, washed with coldaqueous acetic acid, then diethyl ether and dried under vacuum overphosphorus pentoxide to give6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid (1.80 g, 97%). δ_(H)(200 MHz, DMSO-d₆) 1.50-1.80 (6H, m), 2.25 (2H, t), 4.40 (2H, app.t),7.78 (2H, d), 7.92 (2H, dd) and 8.3 (2H, d).

[0189] 3.2O-(N-Succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate

[0190] 6-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic acid (467 mg, 1.0mmol) and O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TSTU; 301 mg, 1.0 mmol) were dissolved inN,N-dimethylformamide (5 ml). To the resulting solution was addedN,N-diisopropylethylamine (183 μl, 1.05 mmol). After leaving to standovernight, the solvent was evaporated under vacuum. The residue waspurified by flash chromatography (silica. 10% ethylacetate/dichloromethane) to giveO-(N-succinimidyl)-6-(2,7-dibromo-9-oxo-9H-acridin-10-yl)hexanoate (510mg, 91%). δ_(H) (200 MHz, DMSO-d₆) 1.5-1.8 (6H, m), 2.72 (2H, t), 2.83(4H, s), 4.44 (2H, app.t), 7.82 (2H, d), 7.95 (2H, dd) and 8.36 (2H, d).Mass spectrum: (ES+) 563+565+567 (M+H), 585+587+589 (M+Na). Accuratemass: (M+Na)=C₂₃H₂₀Br₂N₂O₅Na, requires 584.9637. Found 584.9608 (−4.9ppm).

4. O-(N-Succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate

[0191]

[0192] 4.1 4-Carboxyacridone

[0193] 2,2′-Iminodibenzoic acid (5.27 g, 20.5 mmol) was mixed withphosphorus oxychloride (20 ml). The resulting pale yellow slurry washeated to boiling. The slurry turned initially bright yellow, thendissolved to give a deep red solution which was intensely yellow at themeniscus. After 2 hrs at reflux, excess solvent was evaporated undervacuum to give a dark oil. This was quenched with ice, then diluted with2.0M aqueous HCl (25 ml) and the resulting dark solution re-heated toboiling. After 20 mins a solid precipitated and the mixture became verythick; another 20 mls of water was then added to allow effectivestirring. After 1.5 hrs, the mixture was allowed to cool to ambienttemperature. The yellow solid was collected by vacuum filtration, washedwell with water, then acetone, and dried under vacuum to give4-carboxyacridone (4.61 g, 94%). λ_(max) (EtOH)=408, 390, 256 nm. δ_(H)(300 MHz, DMSO-d₆)7.24-7.33 (2H, m), 7.67-7.76 (2H, m), 8.17 (1H, d),8.38 (1H, dd), 8.47 (1H, dd) and 11.9 (broad s, partially exch). Massspectrum: (ES+) 240 (M+H), 262 (M+Na). Melting Point>300° C.

[0194] 4.2 6-(9-Oxo-9H-acridin-4-carboxamido)hexanoic acid

[0195] 4-Carboxyacridone (2.15 g, 9 mmol), was mixed withN,N-dimethylformamide (15 m) and N,N-diisopropylethylamine (1.6 ml, 9.2mmol) and stirred under nitrogen to give a deep yellow solution. To thiswas added O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (3.5 g, 9 mmol) and stirring continued for 2 hrs.During this time a thick yellow precipitate formed. 6-Aminohexanoic acid(1.45 g, 11 mmol) was then added and stirring continued overnight.Reverse phase chromatographic analysis (C₁₈) (methanol:water, 80:20)indicated two spots at R_(f)=0.75 and R_(f)=0.55. The reaction mixturewas then poured into 0.25M aqueous HCl (200 ml) and the precipitatedproduct collected by vacuum filtration, washing with more dilute HCl andwater. The still-damp solid was recrystallized from ethanol/water anddried under vacuum over phosphorus pentoxide to give6-(9-oxo-9H-acridin-4-carboxamido)hexanoic acid (1.97 g, 62%). λ_(max)(EtOH)=408, 390, 256 nm. δ_(H) (300 MHz, DMSO-d₆) 1.35-1.65 (6H, m),2.24 (2H, t), 3.40 (2H, t), 7.26-7.39 (2H, m), 7.74-7.77 (2H, m),8.21-8.28 (2H, m), 8.44 (2H, app.d), 9.00 (1H, broad t, amide), 12.02(broad s, D₂O exch.) and 12.49 (broad s, D₂O exch.). Mass spectrum:MALDI-TOF, m/z=353.15, M=353.15 for C₂₀H₂₁N₂O₄. Melting Point=218° C.

[0196] 4.3O-(N-Succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate

[0197] 6-(9-Oxo-9H-acridin-4-carbaxamido)hexanoic acid (352 mg, 1.0mmol) and O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TSTU; 301 mg, 10 mmol) were dissolved inN,N-dimethylformamide (5 ml). To the resulting solution was addedN,N-diisopropylethylamine (183 μl, 1.05 mmol). After leaving to standovernight the solvent was evaporated under vacuum. The residue waspurified by flash chromatography (silica. 10-100% ethylacetate/dichloromethane) to giveO-(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carbaxamido)hexanoate (410 mg,91%). δ_(H) (200 MHz, DMSO-d₆) 1.44-1.75 (6H, m), 2.71 (2H, t), 2.79(4H, s), 3.38 (2H, t), 7.27-7.39 (2H, m), 7.75 (2H, app.d), 8.21-8.28(2H, m), 8.44 (1H, app.d), 9.00 (1H, broad t, amide) and 12.47 (broad s,D₂O exch.). Mass spectrum: (ES+) 450 (M+H), 472 (M+Na). Accurate mass:(M+H)=C₂₄H₂₄N₃O₆ , requires 450.1665. Found 450.1671 (+1.3 ppm).

5. 2-Carboxymethyl-7-chloro-9-oxo-9,10-acridine

[0198]

[0199] 5.1 N-(4-Carboxymethylphenyl)-4-chloro-2-carboxyaniline

[0200] To a 100 ml round bottomed flask was added 2,5-dichlorobenzoicacid (1.9 g, 10 mmol), 4-aminophenylacetic acid (1.5 g, 10 mmol),anhydrous sodium carbonate (3.2 g, 26 mmol), activated copper metalpowder (0.25 g, 4 mmol) and 1-butanol (50 ml). The flask was fitted witha magnetic stirrer bar, water condenser, silica gel guard tube andheated under reflux for 48 hours. TLC (RPC₁₈, Water, 20:methanol, 80)showed the formation of a slower moving component at R_(f) 0.55. Thesolvent was removed under reduced pressure with final drying under highvacuum. The residue was dissolved in 50 ml water and heated to boiling,then charcoal was added and the mixture filtered through celite, washingthrough with a further 25 ml of hot water. This solution was cooled to10° C. in an ice bath and then acidified to pH≈2 with concentratedaqueous HCl. The oil that separated was extracted into chloroform, thesolution dried with anhydrous magnesium sulphate, filtered and thesolvent removed by rotary evaporation to leave a sticky solid.Recrystallization from water/acetic acid gave the title compound (1.07g, 35%). δ_(H) (300 MHz, DMSO-d₆) 3.54 (2H, s), 7.15-7.27 (5H, m), 7.39(1H, dd) 7.81 (1H, d), 9.54 (1H, broad s) and 12.0-13.5 (2H, broad).Mass spectrum: (ES−) 304 (M−H). λ_(max) (EtOH)=292, 364 nm.

[0201] 5.2 2-Carboxymethyl-7-chloro-9-oxo-9,10-acridine

[0202] To a 25 ml round bottomed flask was added the diphenylamine (500mg, 1.64 mmol) and phosphorus oxychloride (5 ml). The flask was fittedwith a magnetic stirrer bar, water condenser, silica gel guard tube andheated under reflux for 1 hour. The excess phosphorus oxychloride wasremoved from the dark brown mixture under vacuum, then a small amount ofice was added followed by 2.0M aqueous HCl (10 ml). The mixture washeated to 100° C. for 1 hour and allowed to cool, before beingevaporated to dryness and dried under vacuum over phosphorus pentoxide.The residue was dissolved in 10% v/v water/methanol and eluted through aSepPak RPC₁₈ (10 g) column with monitoring by TLC. Thefluorescence-containing fractions were combined, evaporated to drynessand dried under vacuum over phosphorus pentoxide.

[0203] This semi-purified material was then further purified bypreparative HPLC (RPC₁₈. Water→methanol gradient). Pure fractions werecombined and evaporated to give2-carboxymethyl-7-chloro-9-oxo-9,10-acridine as a pale yellow solid (19mg, 4%). δ_(H) (300 MHz, DMSO-d₆) 3.72 (2H, s), 7.50 (1H, m), 7.58 (1H,m), 7.65 (1H, m), 7.75 (1H, m), 8.10 (1H, m), 8.14 (1H, m) and 11.9+12.4(2×broad s). Mass spectrum: (ES−) 286+288 (M−H), 242+244 (M−H−CO₂).λ_(max) (EtOH)=260, 390, 408 nm.

6. N-(Succinyl)-2-amino-10H-acridine-9-one

[0204]

[0205] 2-Amino-10H-acridine-9-one (100 mg, 0.48 mM), succinic anhydride(50 mg, 0.5Mm), diisopropylethylamine (90 μl) and dry DMF (1 ml) werestirred together overnight. TLC (C-18 reverse phase plates,water:methanol 1:1) indicated that all the starting material (greenfluorescence under long wavelength UV light) had been converted to afaster running spot, which showed blue fluorescence under longwavelength UV light. The solvent was removed by rotary evaporation, theresidue dissolved in dichloromethane and purified by flash columnchromatography (silica, 0-10% methanol/dichloromethane). Pure fractionsof each were pooled and evaporated to dryness to giveN-(succinyl)-2-amino-10H-acridine-9-one, 147 mg (98%). λ_(max) (H₂O)=399nm. δ_(H) (300 MHz, d₆-DMSO) 2.05 (4H, m), 6.82 (1H, t), 7.15 (2H, m),7.28 (1H, t), 7.53 (1H, d), 7.81(1H, d), 8.09(1H,s). Mass spectrum:(ES+) 311. M.Pt>300° C.

7. 2-Nitroacridone-7-sulphonic acid

[0206]

[0207] 7.1 2-Carboxy-4-nitrodiphenylamine

[0208] 2-Chloro-5-nitrobenzoic acid (15 g, 75 mmol) was mixed with1-butanol (100 ml) and stirred. To the resulting mixture was addedaniline (15 ml, 15.3 g, 165 mmol) and N,N-diisopropylethylamine (28.8ml, 21.3 g, 165 mmol). The resulting light yellow solution was heatedunder reflux for 4 days, during which time the colour changed to deepyellow. TLC (RPC₁₈, Methanol, 80:water, 20. R_(f)=0.75, yellow:R_(f)2-chloro-5-nitrobenzoic acid=0.85).

[0209] The solvent was evaporated under vacuum as much as possible. Theresidual oil was triturated with water acidified with aqueous HCl tomaintain a pH of 2-4. A yellow solid eventually separated. This wascollected by vacuum filtration, washed well with excess water andallowed to suck dry over 15 minutes. The crude product was purified bymixing with acetic acid (150 ml), then heating to boiling and allowingthe resulting mixture to cool slowly to ambient temperature withstirring. The bright yellow solid so obtained was collected by vacuumfiltration, washed with acetic acid followed by excess diethyl ether anddried under vacuum over phosphorus pentoxide to give2-carboxy-4-nitro-diphenylamine (12.02 g, 62%). δ_(H) (200 MHz, DMSO-d₆)7.14 (1H, d), 7.24-7.52 (5H, m), 8.18 (1H, dd), 8.72 (1H, d) and 10.38(1H, broad s).

[0210] 7.2 2-Nitroacridone

[0211] 2-Carboxy-4-nitrodiphenylamine (5.16 g, 20 mmol) was mixed withphosphorus oxychloride (20 ml). The resulting yellow slurry was heatedunder reflux for 3 hrs, during which time the solids dissolved to give adark solution (intensely yellow at the meniscus). The excess phosphorusoxychloride was then evaporated under vacuum; the resulting oil wascarefully quenched with ice before addition of 1.0M aqueous HCl (100ml). The mixture was then heated to boiling, whereupon acetic acid (upto 20 ml) was slowly added down the condenser to aid dispersion of theimmiscible oil. On continued boiling for 1 hour, a yellow slurryresulted. After subsequent cooling, this solid was collected by vacuumfiltration, washed with excess water, then acetone and finally diethylether, before drying under vacuum over phosphorus pentoxide to give2-nitroacridone (4.66 g, 97%). δ_(H) (200 MHz, DMSO-d₆) 7.39 (1H,app.t), 7.60 (1H, d), 7.68 (1H, d), 7.84 (1H, app.t), 8.25 (1H, d), 8.48(1H, dd), 8.98 (1H, d) and 12 38 (1H, broad s).

[0212] 7.3 2-Nitroacridone-7-sulphonic acid

[0213] 2-Nitroacridone (25 mg) was mixed with fuming sulphuric acid(˜20% free SO₃, 2.5 ml) to give a reddish solution. This was heated at100° C. for 90 mins, to give a yellowish solution. After cooling, themixture was quenched dropwise into ice (˜15 g); 6 ml of concentrated HClwere added and the mixture left to stand to precipitate out the product.The resulting pale yellow solid was collected by vacuum filtration,washed with a little 4.0M aqueous HCl, then redissolved in water andfiltered into a clean flask. The water was evaporated under vacuum toleave 2-nitroacridone-7-sulphonic acid as a yellow-brown solid. TLC acid(RPC₁₈, Methanol, 80:water, 20. R_(f)=0.8, yellow. Mass spectrum: (ES+)321 (M+H); accurate mass: (M+H)=C₁₃H₉N₂O₆S requires 321.0181. Found321.0187 (1.8 ppm).

8. 0-(N-Succinimidyl)-6-(2-acetamido)-9-oxo-9H-acridin-10-yl)hexanoate

[0214]

[0215] 8.1 O-Ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate

[0216] 2-Nitroacridone (2.4 g; 10 mmol) was stirred with anhydrousmethyl sulphoxide (25 ml) under a nitrogen atmosphere. After 5 minutes,sodium hydride (60% dispersed in oil, 480 mg; 12 mmol) was added to theyellow solution. Stirring was continued for 90 mins. during which timethe solution turned magenta. Ethyl 6-bromohexanoate (2.67 ml; 12 mmol)was added and stirring continued overnight. The reaction mixture waspoured into water (300 ml) and the yellow precipitate was collected byfiltration, washed with water and dried under vacuum. The solid wasdissolved in dichloromethane and anhydrous magnesium sulphate added tothe solution. After filtration, the solution was evaporated to drynessto leave a yellow-brown solid. The crude product was purified by flashchromatography (silica. 0-5% ethyl acetate/dichloromethane) to give 1.19g (50%) of O-ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate. δ_(H)(200 MHz, DMSO-d₆) 1.2(3H, t), 1.7(6H, m), 2.3(2H, t), 4.05(2H, q),4.55(2H, m), 7.45(1H, m), 7.92(2H, d), 8.03(1H, d), 8.35(1H, d),8.50(1H, dd), 9.03(1H, d).

[0217] 8.2 O-Ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate

[0218] O-Ethyl-6-(2-nitro-9-oxo-9H-acridin-10-yl)hexanoate (1.91 g; 5.0mmol) and ammonium formate (1.58 g; 25 mmol) were dissolved in ethanol(100 ml) to give a yellow solution. The solution was stirred undernitrogen and a catalytic amount of 5% palladium on carbon was added.Stirring was continued for 5 hrs. The solution was then filtered throughcelite and the solvent removed by rotary evaporation. The residue wasdissolved in dichloromethane and extracted with water. The organic layerwas dried with anhydrous magnesium sulphate, filtered and the solventremoved by rotary evaporation. The crude product was purified by flashchromatography (silica. 4-6% methanol/dichloromethane) to give 1.66 g(94%) of O-ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate. δ_(H) (200MHz, DMSO-d₆) 1.15(3H, t), 1.6(6H, m), 2.35(2H, t), 4.05(2H, dd),4.4(2H, t), 5.3(2H, s), 7.2(2H, m), 7.6(4H, m), 8.3(1H, d).

[0219] 8.3 6-(2-Amino-9-oxo-9H-acridin-10-yl)hexanoic acid

[0220] O-Ethyl-6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoate (350 mg; 1.0mmol) was dissolved in acetic acid (5 ml) and 1.0M hydrochloric acid (2ml) and refluxed for 4 hrs. The solvent was removed by rotaryevaporation, the residue dissolved in acetic acid and evaporated todryness and the process repeated twice using acetonitrile as solvent.The residue was dried under vacuum to give 370 mg of6-(2-amino-9-oxo-9H-acridin-10-yl)hexanoic acid.

[0221] 8.4 6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid

[0222] 6-(2-Amino-9-oxo-9H-acridin-10-yl)hexanoic acid (370 mg: 1.14mmol) was dissolved in anhydrous pyridine (10 ml) and acetic anhydride(100 μl) followed by diisopropylethylamine (350 μl). The mixture wasstirred for 3 hours. The solution was evaporated to dryness under vacuumand the gummy residue dissolved in dichloromethane. The solution waswashed with 1.0M hydrochloric acid and then brine. The organic phase wasdried with anhydrous magnesium sulphate, filtered and the solventremoved by rotary evaporation to leave a sticky solid. Trituration withether gave a solid which was dried under vacuum to give 360 mg (86%) of6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid.

[0223] 8.50-(N-Succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate

[0224] 6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic acid (360 mg; 1.0mmol) and O-(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (350 mg; 1.0 mmol) were dissolved in anhydrousdimethylformamide (5 ml) and diisopropylethylamine (183 μl) added. Theyellow solution was stirred under nitrogen for 1 hour. The solvent wasremoved by rotary evaporation to leave a brown gum. This was purified byflash chromatography (silica. 10% methanol/ethyl acetate) to give 330 mg(50%) ofO-(N-succinimidyl)-6-(2-acetamido-9-oxo-9H-acridin-10-yl)hexanoate.δ_(H) (200 MHz, DMSO-d₆) 1.8(6H, m), 2.1(3H, s), 2.9(6H, m), 4.5(2H, m),7.3(1H, m), 7.8(3H, m), 8.05(1H, d), 8.35(1H, d), 8.6(1H, s), 10.2(1H,s). Accurate mass. (M+H)=C₂₅H₂₆N₃O₆, requires 464.1822. Found 464.1798(5.1 ppm).

9. O-(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate

[0225]

[0226] 9.1 6-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid

[0227] O-Ethyl-6-(9-oxo-9H-acridin-10-yl)hexanoate (2.0 g; 6.0 mmol) wasdissolved in conc. sulphuric acid (10ml) and the solution heated to 120°C. for 20 hrs. The mixture was allowed to cool and added to ˜50 gm ofcrushed ice. The precipitate was collected by centrifugation, washedwith 3.0M hydrochloric acid and dried under vacuum and over phosphorouspentoxide to give 2.1 g (90%) of6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200 MHz,DMSO-d₆) 1.7(6H, m), 2.26(2H, t ), 4.5(2H, t), 7.37(1H, m), 7.9(5H, m),8.37(1H,d), 8.58(1H, d). Mass spectrum: (ES+) (M+H) 390.

[0228] 9.2O-(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate

[0229] 6-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid (100 mg; 0.25mmol) was dissolved in anhydrous dimethylformamide (3 ml) and evaporatedto dryness on a rotary evaporator. The process was repeated to removetraces of water. O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (90 mg; 0.3 mmol) was added and the mixture dissolvedin anhydrous dimethylformamide (2 ml) and diisopropylethylamine (90 μl).The yellow solution was stirred under nitrogen for 60 mins when TLC(RP₁₈ 50:50 water:methanol) showed all the starting material had beenconverted to a slower moving component. The solvent was removed byrotary evaporation with final drying under high vacuum. No furtherattempts were made to purify this material.

10. 6-(2-Bromo-7-sulpho-9-oxo-9H-acridin-10-yl)hexanoic acid

[0230]

[0231] O-ethyl-6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoate (2.08 gm; 5.0mmol) was dissolved in conc. sulphuric acid (10 ml) and heated to 120°C. for 20 hrs. The mixture was allowed to cool and added to ˜50 gm ofcrushed ice. The precipitate was collected by centrifugation, washedwith 3.0M hydrochloric acid and dried under vacuum in the presence ofphosphorous pentoxide to give 2.2 g (94%) of6-(2-bromo-7-sulpho-9-oxo-9H-acridin-10-yl)hexa noic acid. δ_(H) (200MHz, DMSO-d₆) 1.7(6H, m), 2.25(2H, t), 4.5(2H, m ), 7.9(4H, m), 8.42(1H, d), 8.57(1H, d). Mass spectrum (ES+) (M+H) 468, 470.

11. N-(Maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide

[0232]

[0233] 11.1 N-(Aminoethyl)maleimide hydrochloride

[0234] N-Butoxycarbonyl-2-(aminoethyl)maleimide (200 mg; 0.83 mmol) wasstirred under nitrogen with 4M hydrochloric acid in dioxan (4 ml). Awhite precipitate started to form after a few minutes. Stirring wascontinued for 40 minutes and then the solvent was removed by rotaryevaporation. The resultant white solid was dried under vacuum to give180 mg (100%) of N-(aminoethyl)maleimide hydrochloride. δ_(H) (200 MHz,CD₃OD) 1.38(2H, s), 3.14(2H, t), 3.81(2H, t), 6.90(2H, s).

[0235] 11.2 N-(Maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide

[0236] O-(N-Succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (102 mg;0.25 mmol) was dissolved in anhydrous dimethyl formamide (800 μl) anddiisopropylethylamine (53 μl) added. N-(Aminoethyl)maleimidehydrochloride (53 mg; 0.30 mmol) was added to the yellow solution whichwas left to stand overnight. The solvent was removed by rotaryevaporation and the residue purified by flash chromatography (silica. 2%methanol/dichloromethane). After removal of the solvent by rotaryevaporation a yellow oil remained which slowly crystallised. Triturationwith diethyl ether completed the crystallisation. This material wasfurther purified by preparative TLC (silica. 5%methanol/dichloromethane) extracting the required material with 10%methanol/dichloromethane. Solvent was removed under vacuum, the residuetriturated with ether to give 65 mg (60%) ofN-(maleimido)ethyl-6-(9-oxo-9H-acridin-10-yl)hexanamide. δ_(H) (200 MHz,DMSO-d₆) 1.7(6H, m), 2.02(2H, t), 3.2(2H, dd), 3.45(2H, t), 4.45(2H, t),7.01 (2H, s), 7.35(2H, m), 7.85(5H, m), 8.36(2H, d). Mass spectrum (ES+)(M+H) 432.

12. N-(Maleimido)ethyl-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanamide

[0237]

[0238] O-(N-Succinimidyl)-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanoate(90 mg; 0.15 mmol) was dissolved in anhydrous dimethyl formamide (1.0ml) and diisopropylethylamine (52 μl) added. N-(aminoethyl)maleimidehydrochloride (52 mg; 0.30 mmol) was added to the yellow solution whichwas left to stand overnight. The solvent was removed by rotaryevaporation and the residue purified by HPLC (Vydac RP₁₈semi-preparative column, gradient of water to 25% acetonitrile (bothcontaining 0.1% trifluoroacetic acid) over 30 minutes, detection at 400nm) to give N-(maleimido)ethyl-6-(2-sulpho-9-oxo-9H-acridin-10-yl)hexanamide.

13. 6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid

[0239]

[0240] 13.1 N-(4-Fluorophenyl)anthranilic acid

[0241] 4-Fluoroaniline (1.86 gm; 20 mmol), 2-chlorobenzoic acid (1.56gm; 10 mmol), ethylene glycol (5 ml) and anhydrous sodium carbonate (1.1gm; 10 mmol) were placed in a reaction vessel and stirred untileffervescence ceased. Cupric chloride (100 mg; 0.75 mmol) dissolved in 2ml of water was added to the reaction mixture which was then heated to125° C. for 6 hrs. The reaction was allowed to cool and water (30 ml)and charcoal were added. The mixture was filtered and then acidified topH 2 with conc. hydrochloric acid. The precipitate was collected byfiltration, washed with water and then re-dissolved in 1M sodiumhydroxide solution. Material was re-precipitated by the addition ofacetic acid, filtered off, washed with aqueous acetic acid, then waterand finally dried under vacuum over phosphorous pentoxide to give 862 mg(37%) of N-(4-fluorophenyl)anthranilic acid.

[0242] 13.2 2-Fluoroacridone

[0243] N-(4-Fluorophenyl)anthranilic acid (0.70 gm; 3 mmol) andphosphorous oxychloride (3 ml) were stirred together and heated to 115°C. for 3.5 hours, then allowed to cool. The reaction mixture was placedon ice and small pieces of ice added, a vigorous reaction occurred withthe evolution of hydrogen chloride. When the reaction had subsided,water (15 ml) was added and the mixture was boiled for 2 hours. Oncooling, a solid precipitated out. This was filtered off and washed withwater until the filtrate was colourless. The precipitate was furtherwashed with cold methanol then diethyl ether and dried under vacuum togive 383 mg (59%) of 2-fluoroacridone.

[0244] 13.3 O-Ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate

[0245] 2-Fluoroacridone (213 mg; 1.0 mmol) was dissolved in anhydrousDMF (3 ml) under a nitrogen atmosphere. Sodium hydride dispersed in oil(45 mg; 1.1 mmol) was added and the mixture stirred until effervescenceceased. Ethyl 6-bromoacetate (250μl) was added and the mixture stirredat 70° C. overnight. The solvent was removed by rotary evaporation andthe yellow residue purified by flash chromatography (silica. 4% ethylacetate/dichloromethane) to give 230 mg (65%) ofO-ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate. δ_(H) (200 MHz,DMSO-d₆) 1.20(3H, t), 1.65(6H, m), 2.35(2H, t), 4.05(2H, dd), 4.45(2H,t), 7.35(1H, m), 7.9(5H, m), 8.35(1H, d). Mass spectrum (ES+) (M+H)356.1

[0246] 13.4 6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid

[0247] O-Ethyl-6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoate (71 mg; 0.2mmol) was dissolved in ethanol (2 ml) and 1.0M sodium hydroxide solution(0.4 ml) added and the mixture heated to 90° C. for 90 minutes. Themixture was cooled and water (6 ml) added to give a yellow precipitate.The mixture was cooled on ice and acidified with conc. hydrochloric acidwhen more material precipitated out. The precipitate was filtered off,washed with water then ethanol and dried under vacuum over phosphorouspentoxide to give 47 mg (72%) of6-(2-fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H) (200 MHz,DMSO-d₆) 1.68(6H, m), 2.25(2H, t ), 4.48(2H, t), 7.36(1H, m), 7.85(5H,m), 8.34(1H, d). λ_(max)(ab) 251 nm (ε=45,500/M⁻¹cm⁻¹); 401 nm(ε=7980/M⁻¹cm⁻¹); 421 nm (ε=7980/M⁻¹cm⁻¹). (PBS buffer). λ_(max) (em)434 nm (PBS buffer)

14. 6-(2-Methoxy-9-oxo-9H-acridin-10 -yl)hexanoic acid

[0248]

[0249] 14.1 N-(4-Methoxyphenyl)anthranilic acid

[0250] 4-Methoxyaniline (1.86 gm; 20 mmol), 2-chlorobenzoic acid (1.56gm; 10 mmol), ethylene glycol (5 ml) and anhydrous sodium carbonate (1.1gm; 10 mmol) were placed in a reaction vessel and stirred untileffervescence ceased. Cupric chloride (100 mg; 0.75 mmol) dissolved in 2ml of water was added to the reaction mixture which was then heated to125° C. for 6 hours. The reaction was allowed to cool then water (30 ml)and charcoal were added. The mixture was filtered and acidified to pH 2with conc. hydrochloric acid. The precipitate was collected byfiltration, washed with water and then re-dissolved in 1M sodiumhydroxide solution. Material was re-precipitated by the addition ofacetic acid, filtered off, washed with aqueous acetic acid, then waterand finally dried under vacuum over phosphorous pentoxide to give 1200mg (49%) of N-(4-methoxyphenyl)anthranilic acid. Mass spectrum (ES+)(M+H) 242

[0251] 14.2 2-Methoxyacridone

[0252] Polyphosphoric acid (50 gm) was heated to 160° C. under anitrogen atmosphere. N-(4-methoxyphenyl)anthranilic acid (4.89 gm; 20mmol) was added and the mixture stirred at 160° C. for 15 minutes. Thereaction was cooled rapidly in an ice bath and water added to give agreenish yellow precipitate. This was filtered off, washed with water,then dilute sodium bicarbonate solution and again with water. The solidwas finally dried at 50° C. under vacuum to give 3.67 gm (81%) of2-methoxyacridone. δ_(H) (200 MHz, DMSO-d₆) 3.86(3H, s), 7.23(1H, t),7.55(5H, m), 8.22(1H, d), 11.7(1H, s).

[0253] 14.3 O-Ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate

[0254] 2-Methoxyacridone (2.25 g; 10 mmol) was stirred with anhydrousdimethyl formamide (15 ml) under a nitrogen atmosphere. After 5 minutes,sodium hydride (60% dispersed in oil, 250 mg; 12 mmol) was added and themixture stirred until effervescence ceased. A second lot of sodiumhydride (230 mg) was added and stirring continued until effervescenceceased. Ethyl 6-bromohexanoate (2.67 ml; 15 mmol) was added to theyellow solution and stirring was continued overnight. The reactionmixture was poured into water (300 ml) and the mixture extracted withdichloromethane. The organic phase was washed with 1.0M hydrochloricacid (2×150 ml) then dried over anhydrous magnesium sulphate. Afterfiltration, the solvent was removed by rotary evaporation to give a darkcoloured oil. This was purified by flash chromatography (silica. 5%ethanol/dichloromethane) to give a yellow oil which crystallised ontrituration with diethyl ether/hexane to give 0.83 g (23%) ofO-ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate. Mass spectrum(ES+) (M+H) 367 (M+Na) 389.

[0255] 14.4 6-(2-Methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid

[0256] O-Ethyl-6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoate (367 mg;1.0 mmol) was dissolved in ethanol (10 ml) and 1.0M sodium hydroxidesolution (2.0 ml) added and the mixture heated to 90° C. for 1 hour. Themixture was cooled and water (20 ml) added. The mixture was cooled onice and acidified with conc. hydrochloric acid when a yellow oilseparated. The oil slowly crystallised to a bright yellow solid. Thiswas filtered off, washed with water and dried under vacuum to give 327mg (96%) of 6-(2-methoxy-9-oxo-9H-acridin-10-yl)hexanoic acid. δ_(H)(200 MHz, DMSO-d₆) 1.7(6H, m), 2.25(2H, t), 3.9(3H, s), 4.5(2H, t), 7.31(1H, m), 7.48(1H, dd), 7.82(4H, m), 8.35(1H, d). λ_(max)(ab) 255 nm(ε=38,100/M⁻¹cm⁻¹); 408 nm (ε=7150/M³¹ ¹cm⁻¹); 428 nm (ε=7150/M⁻¹cm⁻¹).(PBS buffer). λ_(max)(em) 467 nm. (PBS buffer).

15. Fluorescence Lifetime Studies

[0257] Fluorescence lifetimes were determined by time-correlated singlephoton counting using an Edinburgh Instruments FL900CDT Time ResolvedT-Geometry Fluorimeter. Samples were excited at 400 nm using a hydrogenarc lamp. Detection was at 450 nm. Deconvolution using a non-linearleast squares algorithm gave the results shown in Table 2. FIG. 2 is aplot showing the fluorescence lifetimes of certain acridone dyesaccording to the invention. TABLE 2 Fluorescence Lifetimes LifetimeCompound Solvent (nsecs) N-(Succinyl)-2-amino-10H-acridine-9-one water17.2 2-Carboxymethyl-7-chloro-9-oxo-9,10-acridine MeOH 16.86-(2,7-Disulphonato-9-oxo-9H-acridin-10- water 14.6 yl)hexanoic acid6-(9-Oxo-9H-acridin-10-yl)hexanoic acid water/ 14.2 MeOH 50/50)6-(2-Bromo-9-oxo-9H-acridin-10-yl)hexanoic MeOH 8.3 acid6-(2,7-Dibromo-9-oxo-9H-acridin-10-yl)hexanoic MeOH 4.5 acid6-(9-Oxo-9H-acridin-4-carboxamido)hexanoic water/ 4.2 acid MeOH (50/50)2-Nitroacridone-7-sulphonic acid water non- fluorescent6-(2-Acetamido-9-oxo-9H-acridin-10-yl)hexanoic water 17 acid6-(2-Sulpho-9-oxo-9H-acridin-10-yl)hexanoic water 13.3 acid6-(2-Bromo-7-sulpho-9-oxo-9H-acridin-10- water 5.6 yl)hexanoic acid6-(2-Fluoro-9-oxo-9H-acridin-10-yl)hexanoic acid water 146-(2-Methoxy-9-oxo-9H-acridin-10-yl)hexanoic water 17 acid

16. Protein Labelling 1399

[0258] 16.1 Preparation of 6-(9-oxo-9H-acridin-10-yl)hexanoicacid—bovine Serum Albumin (BSA) Conjugate (Conjugate 1)

[0259] To 10 ml of bovine serum albumin (1 mg/ml in 0.1M carbonatebuffer, pH9.3), 100 μlO-(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (1 mg/10 μl inDMSO) was added dropwise whilst stirring. Gentle stirring continued for1 hr at ambient temperature in a foil wrapped vial. Unconjugated dye wasremoved by overnight dialysis (12-14K MWCO) at 4° C. with at least 2changes of PBS. Conjugate 1 was recovered and stored at 4° C.

[0260] 16.2 Preparation of 6-(9-oxo-9H-acridin-4-carboxamido)hexanoicacid-rabbit serum albumin conjugate (Conjugate 2)

[0261] To 10 ml of rabbit serum albumin (1 mg/ml in 0.1M carbonatebuffer, pH9.3), 100μO-(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate (1mg/100 μl in DMSO) was added dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial.Unconjugated dye was removed by overnight dialysis (12-14K MWCO) at 4°C. with at least 2 changes of PBS. Conjugate 2 was recovered and storedat 4° C.

[0262] 16.3 Determination of the Fluorescence Lifetimes of Conjugates 1and 2

[0263] The fluorescence lifetimes of a mixture of conjugates 1 and 2were determined in PBS. The results are shown in FIG. 3. Deconvolutionand curve fitting using a non-linear least-squares algorithm gave theresults shown in Table 3. TABLE 3 Lifetime Relative Sample (ns) %Conjugate 1 14.0 40.2 5.5 45.6 1.2 14.3 Conjugate 2 4.6 49.4 1.2 35.019.3 15.6

[0264] 16.4 Immunoprecipitation Assay

[0265] To 500 μl of PBS in a 1.5 ml centrifuge tube was added 200 μlconjugate 1 and 260 μl conjugate 2. After mixing, 450 μl was removedinto a silica cuvette for determination of the fluorescence lifetimeusing excitation at 405 nm, emission at 450 nm by a time-correlatedsingle photon counting technique (Edinburgh Analytical InstrumentsFL900CDT spectrometer). To the 450 μl in the centrifuge tube, 100 μl ofanti-BSA antibody was added. After incubation for 30 min at 37° C., then1 hr incubation at 4° C., the tube was centrifuged for 5 min in abench-top centrifuge. The pellet was washed twice with ice-cold PBS,then re-suspended in 0.1M NaOH. The fluorescence lifetime of thissolution was determined as above. The results are shown in FIG. 4.Deconvolution and curve fitting using a non-linear least-squaresanalysis algorithm gave the results shown in Table 4. TABLE 4 LifetimeFits for Immunoprecipitation Assay Lifetime Relative Sample (ns) %Initial mixture 14.7 33.3 5.0 47.5 0.9 19.2 Re-suspended pellet 13.170.3 4.7 22.8 0.7 6.9

[0266] The results show that the relative percentage of conjugate 1(lifetime range 13-15 ns) has increased significantly in the pellet, asa result of the immunoprecipitation by the anti-BSA antibody. Theproportion of conjugate 2 (lifetime range 4.5-5 ns) in the re-suspendedpellet is correspondingly decreased, relative to the proportion in theinitial mixture. Although the immunoprecipitation process was notcompletely efficient, analysis using fluorescence lifetime has enabledthe resolution of two species (conjugate 1 and conjugate 2) in mixtureswhich are indistinguishable by their emission wavelength.

17. Fluorescence Lifetime Detection in Capillary Electrophoresis ofAcridone Dye-labelled DNA Fragments

[0267] M13 DNA primers were labelled using standard techniques with eachof four acridone dyes according to the present invention, i.e:

[0268] i) 6-(2-(acetylamino)-9-oxo-9H-acridin-10-yl)hexanoic acid,

[0269] ii) 6-(9-oxo-9H-acridin-10-yl)hexanoic acid,

[0270] iii) 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid, and

[0271] iv) 6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid.

[0272] The succinimidyl ester of each dye was conjugated to anamine-modified M13 forward sequencing primer in 0.1M carbonate pH9.3/DMF (final composition 2:1). Purification by HPLC on a C18 columnused a triethylammonium bicarbonate/MeCN solvent system.

[0273] Real-time fluorescence lifetime detection was achieved byinterfacing a commercial multiharmonic Fourier transform (MHF)fluorescence lifetime instrument (Model 4850MHF, SpectronicsInstruments, Rochester, N.Y.) to a Beckman P/ACE 5000 CE system (Li, L.et al, J. Chromatogr. B, (1997), 695, 85-92). The excitation source wasa continuous wave violet diode laser that supplied 25-30 nW at 405 nm.The laser beam was focused onto the detection window of the capillaryusing either a 45 mm focusing lens or a 6.3× microscope objective with afocal length of 22 mm. The emission signal was collected by a 40×microscope objective. Emission was selected through a 435 nm long passfilter. A cross-correlation frequency of 9.4 Hz was used in the lifetimemeasurements, resulting in 9.4 phase and modulation measurements persecond. Ten successive measurements were then averaged prior to dataanalysis to yield approximately one lifetime measurement per second.Scattered light from the capillary provided the lifetime reference.

[0274] Solutions of 0.5 mM dye-labelled primer in 100 mM Tris buffer, pH8.6, were injected into the bare 75 μM internal diameter capillary for10 s each at 10 kV injection voltage. The separation voltage was 18 kV(250 V/cm). The replaceable gel matrix contained 2% POP-6 gel in3.5×POP-6 buffer. The lifetime electropherogram as shown in FIG. 5 wasobtained for successive injections of M13 DNA primers labelled with eachof the four dyes. The solid line is the intensity and the dotscorrespond to lifetimes recovered from a 1-component fit usingnon-linear least squares analysis software (Globals, Inc). The resultsshow that the fluorescence lifetime (dots) coincides with thefluorescence intensity peaks (line) as the dye labelled M13 primersmigrate past the detector.

18. Multiplexing Fluorescence Lifetime Determination

[0275] The following fluorescent acridone dye derivatives were preparedas 1 mg/ml stock solutions in methanol:

[0276] a) 6-(9-oxo-9H-acridin-10-yl)hexanoic acid;

[0277] b) 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic acid; and

[0278] c) 6-(9-oxo-9H-acridin-4-carboxamido) hexanoic acid.

[0279] The methanolic stock solutions were diluted (1/100) into 12%polyacrylamide mix which was allowed to polymerize directly indisposable cuvettes. Mixtures of the dyes were similarly prepared. Thefluorescence lifetimes of samples containing single, or mixtures of dyesin polyacrylamide gel, were recorded by a time-correlated single photoncounting technique (Edinburgh Analytical Instruments FL900CDTspectrometer). Samples were excited at 400 nm using a hydrogen arc lamp,detection was at 450 nm. Deconvolution using a non-linear-least squaresalgorithm gave the results shown in Table 5. TABLE 5 Principal LifetimeRelative Sample (ns) % a) 6-(9-oxo-9H-acridin-10-Yl)hexanoic acid 14.096.8 b) 6-(2-bromo-9-oxo-9H-acridin-10-yl)hexanoic 9.0 77.1 acid c)6-(9-oxo-9H-acridin-4-carboxamido)hexanoic 3.6 74.4 acid a) + c) 14.074.5 3.9 25.5 b) + c) 9.5 49.7 4.0 50.3 a) + b) + c) 14.3 55.5 7.9 26.73.5 17.9

[0280] The results show that multiple lifetime components are reportedwhen the dyes are analysed in PAGE. However, the principal lifetimecomponent of each of the single dyes can still be distinguished inmixtures of the dyes in PAGE. Thus, with prior knowledge of the lifetimecomponents present, the potential to multiplex fluorescence lifetimeshas been demonstrated.

19. Co-localisation of Bovine Serum Albumin (BSA) Labelled withDifferent Acridone Dyes using SDS PAGE

[0281] 19.1 Preparation of 6-(9-oxo-9H-acridin-4-carboxamido)hexanoicacid—bovine serum albumin (BSA) conjugate (Conjugate 3)

[0282] To 1 ml of BSA (10.0 mg/ml in 0.1M NaHCO₃ solution) was added asolution ofO-(N-succinimidyl)-6-(9-oxo-9H-acridin-4-carboxamido)hexanoate (lifetime4 ns) (25 μl; 0.3125 mg/ml in DMSO). The resulting mixture was incubatedat room temperature for 30 minutes with occasional mixing. A PD10 column(Amersham Biosciences) was equilibrated with 10 ml of phosphate bufferedsaline (PBS; pH 7.4). The dye-labelled BSA was added to the column, thecolumn washed with PBS (2 ml) and then eluted with 3 ml of PBS. Theeluate was collected to yield Conjugate 3.

[0283] 19.2 Preparation of 6-(9-oxo-9H-acridin-10-yl)hexanoicacid—bovine Serum Albumin (BSA) Conjugate (Conjugate 4)

[0284] To 1 ml of BSA (10.0 mg/ml in 0.1M NaHCO₃) was added a solutionof O-(N-succinimidyl)-6-(9-oxo-9H-acridin-10-yl)hexanoate (lifetime 14ns) (2 mg in 100μl of DMSO). The resulting mixture was incubated at roomtemperature for 30 minutes with occasional mixing. A PD10 column(Amersham Biosciences) was equilibrated with 10 ml of phosphate bufferedsaline (PBS; pH 7.4). The dye-labelled BSA was added to the column, thecolumn washed with PBS (2 ml) and then eluted with 3 ml of PBS. Theeluate was collected to yield Conjugate 4.

[0285] 19.3 Sample Preparation and Electrophoresis

[0286] Conjugates 3 and 4 prepared as above, were mixed together in aratio of 2:1 in 0.05M Tris (20 μl); buffered to pH 7.5 with acetic acidcontaining 1% w/v sodium dodecyl sulphate, Bromophenol Blue (10 mg/100ml) and dithiothrietol (154 mg/100 ml) (Amersham Biosciences). Proteinsamples were reduced by heating to 95° C. for 3 minutes. Electrophoresiswas performed using a MultiPhor II flat bed electrophoresis system withExcelGel SDS buffer strips (anode strip: 0.45 mol/Tris/acetate pH 6.6, 4g/L SDS and 0.05 g/L Orange G; cathode strip: 0.08 mol/L Tris, 0.80mol/L Tricine and 6.0 g/L SDS pH 7.1). Duplicate samples were applied tothe surface of a pre-formed Excel 8-18 SDS PAGE gradient gel (AmershamBiosciences) using a paper sample application strip placed at locationscorresponding with 96-well microplate centres required for scanning thegel. Molecular weight markers were run in separate lanes, so that partof the gel could be stained using Coomassie Blue to check the integrityof the samples, monitor molecular weight and to orientate the gel forlifetime scanning. Electrophoresis was initiated at constant current toa maximum voltage of 500V for 85 minutes with the flat bed temperaturemaintained at 15° C. Prior to scanning, the gel was fixed in aqueoussolution of 25% methanol, 5% acetic acid v/v for at least 30 minutes.Following lifetime scanning, the gel was stained for 10-20 minutes in0.1% Coomassie Blue G-250 in aqueous solution of 25% methanol, 5% aceticacid v/v and de-stained in aqueous solution of 25% methanol, 5% aceticacid v/v.

[0287] 19.4 Scanning

[0288] Fixed gels were scanned with single wavelength laser excitationat 405 nm and the gel sampled at approximately 2 mm intervals along theaxis of the electrophoretic separation. Data was analysed using aBayesian algorithm to assign fluorescence intensity to fluorescencelifetimes of 2 ns (gel background); 4 ns (4 ns acridone dye); 6 ns(intrinsic BSA lifetime); and 14 ns (14 ns acridone dye).

[0289]FIG. 6 shows the 4 ns dye-labelled BSA and the 1 4 ns dye-labelledBSA (mixed in a ratio of 2:1) and co-electrophoresed in the same gellane. Two peaks are resolved at 4 ns and 14 ns, both corresponding tothe position of BSA relative to molecular weight markers. The twolabelled BSA species are co-located but distinguishable by lifetimediscriminated intensity. Both BSA species are resolved by the gel to thesame location in the gel as indicated by reference to molecular weightmarkers and post electrophoresis staining (66 kD).

1-29. (cancelled)
 30. In a process for labelling and lifetime detectionof a target material, the improvement comprising labeling with a dye ofthe formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete a one ring, atwo fused ring, or a three fused ring aromatic or heteroaromatic system,wherein each ring includes five or six atoms selected from carbon atomsand 0-2 atoms selected from oxygen, nitrogen and sulphur; R², R³, R⁴ andR⁵ are independently selected from hydrogen, halogen, amide, hydroxyl,cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group -E-Fand the group —(CH₂—)_(n)Y; R¹ is selected from hydrogen, mono- ordi-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl, the group -E-F andthe group —(CH₂—)_(n)Y; E is a spacer group having a chain from 1-60atoms selected from the group consisting of carbon, nitrogen, oxygen,sulphur and phosphorus atoms and F is a target bonding group; Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6; And furtherwherein at least one of groups R², R³, R⁴ and R⁵ is a water solubilisinggroup selected from sulphonate, sulphate, quaternary ammonium and thegroup —(CH₂—)_(n)Y; and/or R¹ is the group —(CH₂—)_(n)Y.
 31. The methodof claim 30, wherein said dye is a fluorescent dye wherein: groups R²and R³ are attached to the Z¹ ring structure and groups R⁴ and R⁵ areattached to the Z² ring structure; R², R³, R⁴ and R⁵ are independentlyselected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- ordi-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group -E-Fand the group —(CH₂—)_(n)Y; and R¹ is selected from hydrogen, C₁-C₂₀alkyl, aralkyl, the group -E-F and the group —(CH₂—)_(n)Y.
 32. Themethod of claim 31, wherein said fluorescent dye has a fluorescencelifetime in the range from 2 to 30 nanoseconds.
 33. The method of claim30, wherein said dye is a non-fluorescent or substantiallynon-fluorescent dye wherein at least one of groups R¹, R², R³, R⁴ and R⁵includes at least one nitro group.
 34. The method of claim 30, whereinat least one of groups R¹, R², R³, R⁴ and R⁵ is the group -E-F.
 35. Themethod of claim 30, wherein said target bonding group F includes areactive group for reacting with a functional group on a targetmaterial, or a finctional group for reacting with a reactive group on atarget material.
 36. The method of claim 35, wherein said reactive groupis selected from carboxyl, succinimidyl ester, sulpho-succinimidylester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide,vinylsulphone, dichlorotriazine and phosphoramidite.
 37. The method ofclaim 35, wherein said functional group is selected from hydroxy, amino,sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphateand thiophosphate.
 38. The method of claim 30, wherein said spacer groupE is selected from: —(CHR′)_(p)— —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)——{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)——{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, or phenylene substituted with sulphonate, p is 1-20, q is0-10,r is 1-10and s is 1-5.
 39. A method for labelling a targetbiological material, the method comprising: i) adding to a liquidcontaining said target biological material a dye of formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete a one ring, atwo fused ring, or a three fused ring aromatic or heteroaromatic system,wherein each ring includes five or six atoms selected from carbon atomsand 0-2 atoms selected from oxygen, nitrogen and sulphur; at least oneof the groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where E and F arehereinbefore defined; when any of said groups R², R³, R⁴ and R⁵ is notsaid group -E-F, said remaining groups R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; when group R′ is not said group -E-F, it isselected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; and firther wherein at least one of remaining groups R²,R³, R⁴ and R⁵ is a water solubilising group selected from sulphonate,sulphate, quaternary ammonium and the group —(CH₂—)_(n)Y; and/or R¹ isthe group —(CH₂—)_(n)Y, where Y and n are hereinbefore defined; and ii)incubating said dye with said target biological material underconditions suitable for labelling said target.
 40. The method of claim39, wherein said target biological material is selected from the groupconsisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan arnino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs, hormones, cells, cell membranes and toxins.41. A method for the assay of an analyte in a sample which methodcomprises: i) contacting the analyte with a specific binding partner forsaid analyte under conditions suitable to cause the binding of at leasta portion of said analyte to said specific binding partner to form acomplex and wherein one of said analyte and said specific bindingpartner is labelled with a fluorescent dye of formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete a one ring, atwo fused ring, or a three fused ring aromatic or heteroaromatic system,wherein each ring includes five or six atoms selected from carbon atomsand 0-2 atoms selected from oxygen, nitrogen and sulphur; at least oneof groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where E is a spacergroup having a chain from 1-60 atoms selected from the group consistingof carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is atarget bonding group; when any of said groups R², R³, R⁴ and R⁵ is notsaid group -E-F, said remaining groups R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when group R¹ is not said group -E-F,it is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; and further wherein at least one of remaining groups R²,R³, R⁴ and R⁵ is a water solubilising group selected from sulphonate,sulphate, quaternary ammonium and the group —(CH₂—)_(n)Y; and/or R¹ isthe group —(CH₂—)_(n)Y, where Y and n are hereinbefore defined; ii)measuring the emitted fluorescence of the labelled complex; and iii)correlating the emitted fluorescence with the presence or the amount ofsaid analyte in said sample.
 42. The method of claim 41, wherein saidanalyte-specific binding partner is selected from the group consistingantibodies/antigens, lectins/glycoproteins, biotin/streptavidin,honnone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA andDNA/binding protein.
 43. A set of two or more different fluorescentdyes, each dye of said set of dyes having the formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete a one ring, atwo fused ring, or a three fused ring aromatic or heteroaromatic system,wherein each ring includes five or six atoms selected from carbon atomsand 0-2 atoms selected from oxygen, nitrogen and sulphur; at least oneof groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where E is a spacergroup having a chain from 1-60 atoms selected from the group consistingof carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is atarget bonding group; when any of said groups R², R³, R⁴ and R⁵ is notsaid group -E-F, said remaining groups R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl, carbonyl,carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl,C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammoniumand the group —(CH₂—)_(n)Y; and, when group R¹ is not said group -E-F,it is selected from hydrogen, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; and further wherein at least one of remaining groups R²,R³, R⁴ and R⁵ is a water solubilising group selected from sulphonate,sulphate, quaternary ammonium and the group —(CH₂—)_(n)Y; and/or R¹ isthe group —(CH₂—)_(n)Y, where Y and n are hereinbefore defined; andwherein each dye of said set has a distinguishably differentfluorescence lifetime compared with the lifetimes of the remaining dyesof the set.
 44. The set of claim 43 including four different dyes, eachdye of the set having a different fluorescence lifetime.
 45. The set ofclaim 43, wherein each of the fluorescent dyes in the set has afluorescence lifetime in the range from 2 to 30 nanoseconds.
 46. The setof claim 43, wherein the difference in the lifetimes of the fluorescentemission of at least two dyes is at least 15% of the value of theshorter lifetime dye.
 47. The method of claim 33, wherein thefluorescence of said dye is enhanced by reducing at least one nitrogroup to —NHOH or —NH₂
 48. The method of claim 33, for detectingnitroreductase enzyme activity in a composition further comprising: i)mixing under conditions to promote nitroreductase activity saidcomposition with the dye; and ii) measuring an increase in fluorescencewherein said increase is a measure of the amount of nitroreductaseactivity.
 49. The method of claim 33, further comprising: i) binding onecomponent of a specific binding pair to a surface; ii) adding a secondcomponent of the specific binding pair under conditions to promotebinding between the components, said second component being labelledwith a nitroreductase enzyme; iii) adding the dye; and iv) detectingbinding of the second component to the first component by measuring anincrease in fluorescence as a measure of bound nitroreductase activity.50. The method of claim 49, wherein said specific binding pair isselected from the group consisting of antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hornone/receptor,enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.
 51. Themethod of claim 33, further comprising: i) contacting a host cell whichhas been transfected with a nucleic acid molecule comprising expressioncontrol sequences operably linked to a sequence encoding anitroreductase, with the dye; and ii) measuring an increase influorescence as a measure of nitroreductase gene expression.
 52. Themethod of claim 51, wherein said method is conducted in the presence ofa test agent whose effect on gene expression is to be determined.
 53. Adye of the formula:

wherein: groups R² and R³ are attached to the Z¹ ring structure andgroups R⁴ and R⁵ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete one ring, twofused ring, or three fused ring aromatic or heteroaromatic systems,wherein each ring includes five or six atoms selected from carbon atomsand 0-2 atoms selected from oxygen, nitrogen and sulphur; at least oneof groups R¹, R², R³, R⁴ and R⁵ is the group -E-F where E is a spacergroup having a chain from 1-60 atoms selected from the group consistingof carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is atarget bonding group; and, when any of said groups R¹, R², R³, R⁴ and R⁵is not said group -E-F, said remaining groups R², R³, R⁴ and R⁵ areindependently selected from hydrogen, halogen, amide, hydroxyl, cyano,nitro, amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl,carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl,heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,quaternary ammonium and the group —(CH₂—)_(n)Y; and, when group R¹ isnot said group -E-F, it is selected from hydrogen, mono- ordi-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl and the group—(CH₂—)_(n)Y; Y is selected from sulphonate, sulphate, phosphonate,phosphate, quaternary ammonium and carboxyl; and n is an integer from 1to 6; and further wherein at least one of remaining groups R², R³, R⁴and R⁵ is a water solubilising group selected from sulphonate, sulphate,quaternary ammonium and the group —(CH₂—)_(n)Y; and/or R¹ is the group—(CH₂—)_(n)Y, where Y and n are hereinbefore defined.
 54. The dye ofclaim 53, wherein said target bonding group F comprises: i) a reactivegroup selected from carboxyl, succinimidyl ester, sulpho-succinimidylester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide,vinylsulphone, dichlorotriazine and phosphoramidite; or ii) a functionalgroup selected from hydroxy, amino, sulphydryl, imidazole, carbonylincluding aldehyde and ketone, phosphate and thiophosphate.
 55. The dyeof claim 53, wherein said spacer group E is selected from: —(CHR′)_(p)——{(CHR′)_(q)—O—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—NR′—(CHR′)}_(s)——{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, optionally substituted with sulphonate, p is 1-20, q is0-10, r is 1-10 and s is 1-5.